Theoretical Insights into the Reaction and Inhibition Mechanism of

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Theoretical Insights into the Reaction and Inhibition Mechanism of Metal-Independent Retaining Glycosyltransferase Responsible for Mycothiol Biosynthesis Juan I. Blanco Capurro, Chad W. Hopkins, Gustavo Pierdominici-Sottile, Mariano Camilo Gonzalez Lebrero, Adrian E. Roitberg, and Marcelo A. Marti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10130 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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The Journal of Physical Chemistry

Theoretical Insights into the Reaction and Inhibition Mechanism of Metal-Independent Retaining Glycosyltransferase Responsible for Mycothiol Biosynthesis Juan I. Blanco Capurro1,2, Chad W. Hopkins3, Gustavo Pierdominici Sottile4, Mariano C. González Lebrero5, Adrian E. Roitberg6 * and Marcelo A. Marti1,2 * 1- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina. 2- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN) CONICET, Ciudad Universitaria, Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina. 3- Department of Physics, University of Florida, Gainesville, Florida 32611, United States. 4- Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Sáenz Pe ña 352, Bernal B1876BXD, Argentina. 5- INQUIMAE-DQIAQF, Facultad de Ciencias Exactas y Naturales, UBA and CONICET. 6- Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States.

*) To whom correspondence should be sent: [email protected] , [email protected]

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ABSTRACT Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center and, therefore, they are classified as either inverting or retaining GTs. While inverting GTs reaction mechanism has been widely studied, retaining GTs mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNAN in IUPAC nomenclature) and has a 17.5 kcal/mol Free Energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion like Transition State. In contrast, fluorinated substrate-analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the Transition State.

INTRODUCTION GycosylTransferases (GTs), EC 2.4.x.y, are ubiquitous enzymes available in all forms of life, that establish natural glycosidic linkages between a carbohydrate and a wide range of molecules, yielding oligo and polysaccharides, glycoproteins, glycolipids and cell wall components, antibiotics, macrolides, and nucleic acids. Generically, they catalyse the transfer of a carbohydrate moiety from an activated donor (the 'glycosyl donor') to a specific acceptor molecule, and whenever the donor is a diphospho sugar-nucleotide derivative (NDP), they are usually called 'Leloir enzymes', in honor to the argentinian nobel laureate1.


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Structurally, GTs are currently classified into more than 90 CAZy families, and they usually adopt one of two folds2. The GT-A fold consists of 2 structurally different domains that yield a rather compact, globular structure, and usually binds divalent cations in the active site, which play a key role in the catalityc reaction by stabilizing the glycosyl donor and aiding in leaving group departure; The GT-B fold consists of two domains with Rossman-type folds connected by a hinge zone, and the active site locates right between the middle of these domains. In GTs, the glycosyl-transfer reaction may proceed with one of the two stereochemical outcomes regarding the configuration of the anomeric center, which may be either inverted or retained. Consequently, and given the fact that different enzymes perform their task with a strict stereochemical control, they have been categorized as either 'inverting' or 'retaining' GTs. Interestingly, the inverting/retaining classification of GTs was shown to be independent of the fold, since both GT-A and GT-B examples of inverting and retaining enzymes have been reported1,2. Inverting GTs have been traditionally the most studied3-7, since inversion of the anomeric configuration often occurs in a straightforward process involving a concerted SN2-like single displacement mechanism (Figure 1A), in which the acceptor molecule attacks the anomeric center from the opposite face of the leaving group. Also, in addition to stabilization of the transition state, inverting enzymes active sites provide a general base function, typically performed by a carboxylate residue, that activates the acceptor nucleophile1. Retaining GTs, on the other hand, have always been controversial. Original models suggested that retention was by analogy with retaining glycosidases - the result of a double inversion, generated by two sequential SN2 (Figure 1B): the first SN2 occurring with an enzyme’s residue acting as a nucleophile that results in the formation of an 'inverted covalent-enzyme intermediate', after which a second SN2 driven by the acceptor moiety would take place to yield the final product restoring the initial configuration. In other words, early proposals of retaining mechanism consisted of two sequential inverting steps. Later on, as increasingly more retaining GT structures began to be reported, the absence of properly located residues that could be responsible for the covalent-enzyme intermediates in many active sites led - as previously proposed for glycogen phosphorilase10 - to the so called "SNi-like" mechanism model (also referred in literature as "front-side” or “single displacement")1,8-11. In this model, the nucleophilic attack and leaving group departure occur in the same face of the donor sugar through an oxocarbenium ion-like transition state (Figure 1C) or intermediate (Figure 1D), and it is the NDP diphosphate group itself that acts as the base that deprotonates the acceptor. Given their ubiquity and relevance, understanding GTs mechanism is an active field of research. Moreover, unveiling GT catalyzed reaction mechanisms with atomic detail is a crucial aspect to understand the microscopic origin of their catalytic efficiency, with a potential impact in protein engineering and drug design projects, since they allow not only to identify key residues involved in the reaction and substrate recognition process, but also the rational design of novel inhibitors that, for example, mimic transition state structures12,13. In this context, computational methods offer both a powerful predictive tool and an invaluable complement to experimental data.


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Fig 1. Various Reaction Mechanisms proposed for GTs: A) Inverting Mechanism; B) Double-Displacement Retaining Mechanism; C) SNi-like concerted Retaining Mechanism (ANDN); D) SNi-like step-wise Retaining Mechanism (DN *ANss ).

Density Functional Theory (DFT) based hybrid quantum mechanical/molecular mechanical (QM-MM) methods are among the best choices for studying enzymatic reaction mechanisms, since they allow to get an accurate QM description of the chemical events that take place in the enzyme's active site (breaking/forming of chemical bonds and charge fluctuation as a function of geometry), while treating the rest of the system and its environment using a less computational-expensive classically-derived force field. We have a long experience in using these type of methods to describe enzymatic reaction mechanisms14-19, and we have recently implemented a pure DFT method -using the Perdew Burke and Ernzerhof (PBE) exchange correlation functional- in the popular AMBER package which runs on powerful Graphical Processing Units (GPU)20,21, allowing the user to determine free energy profiles (i.e including entropic and dynamic contributions) of challenging reactions as those presented by GTs. So far, the SNi-like mechanism has been successfully modeled by several groups in both GT-A and GT-B examples of retaining enzymes using computer simulations with different levels of theory and sampling schemes22-24. However, despite accurately and consistently describing the SNi-like mechanism, some cases have been shown to favor a highly dissociative mechanism with an oxocarbenium-ion-like transition state25 (IUPAC ANDN ,Figure 1C), while others seem to prefer a step-wise mechanism with a short-lived oxocarbenium-ion intermediate26-34 (IUPAC


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AN*DNSS , where the asterisk stands for “short-lived intermediate” and the “ss” stands for “solvent-separeted ion pair”, Figure 1D). Despite some key aspects of the reaction such as the particular order of bond forming and bond breaking events and the nature of the base that deprotonates the attacking -OH have reached general consensus, the debate of whether the front-face mechanism takes place on a single step or as a step-wise reaction is the main question that shows that our understanding of the SNi-like mechanism is still incomplete. Moreover, to our knowledge, there is a lack of studies analyzing how the substrate analogues that act as inhibitors actually prevent the enzyme from performing its task. In this context, and to make our contribution to the field, in the present work we analyzed using our in-house developed QM(DFT-PBE)/MM method the reaction and inhibition mechanism of a key retaining metal-independent GT-B from Actinobacteria, MshA35. Mycothiol (Msh) is a low weight thiol compound found in Actinobacteria -gram positive bacteria-, a phylum that includes pathogens like Mycobacterium tuberculosis and Mycobacterium leprae, whose function is analogous to that of Gluthathione in gram negative bacteria and eukaroytes36. It plays a very important role in the bacillus pathogenicity and its absence renders the bacteria highly sensitive to immune’s system nitrosative attack. Among the enzymes involved in mycothiol biosynthesis, glycosyltransferase ‘MshA’ catalyzes the first step of the metabolic pathway, and has proved to have many key features that make it a potential therapeutic target37-39. MshA (family GT4) is a metal-independent retaining GT-B that catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from donor UDP-GlcNAc to acceptor 1-L-myo-inositol-1-phosphate (m-Ins-1P), to yield 3-phospho-1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins-P)36,37. Based on the available X-ray structure of Corynebacterium glutamicum MshA in complex with UDP and m-Ins-1P (PDB id: 3C4V) and steady state kinetic data35, the following general mechanism was proposed: m-Ins-1P's O3 oxygen performs a nucleophilic attack on GlcNAc's anomeric center (C1) to form the glycosidic bond, and is concomitantly deprotonated by UDP's β-phosphate group (oxygen O3B) which acts as the leaving group. However, several questions remain unanswered, particularly related to the specific order of events, the potential presence of a stable ionic intermediate, and how catalysis is actually achieved. Furthermore, the available kinetic data with the substrate analogue UDP-(5F)-GlcNAc suggest that it acts as a competitive inhibitor40, but interpretation of the data is not straightforward, particularly in relation to how precisely the presence of the fluorine interferes in the glycosyl-transfer reaction. Thus, given the available data, and its relevance for future drug development projects, we strongly believe MshA is an ideal case for molecular simulations studies of the GTs reaction and inhibition mechanisms, and have therefore undertaken its study. Our results, show that MshA works through a concerted SNi-like mechanism, and that the key effect of the fluorine atom in the inhibitor is to prevent charge transfer that compensates the positive charge development in the anomeric carbon during transition state formation.

COMPUTATIONAL METHODS Starting structure and Equilibration protocol Coordinates were retrieved from Protein Data Bank, (PDB id: 3C4V) for Corynebacterium glutamicum MshA in complex with UDP and m-Ins-1P35. Since this initial structure presented two empty gaps (residues 139-145; 266-273), they were completed by superimposition of all chain monomers available (including the ones from the apo-structure, PDB id: 3C48). Final missing residues were added using the Modeller 9.9 software41. The sugar donor, GlcNAc, was added ACS Paragon Plus Environment

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onto this complete model using an in-house developed bias-docking approach, specially designed for docking of carbohydrate molecules42 running on the Autodock 4 software43. The complete ternary complex was then solvated with TIP3P water molecules in an octahedral box. The final solvated system consisted of over 36,000 atoms. All subsequent Molecular Dynamics simulations were performed with the AMBER ff99SB force field44 for the protein and the GLYCAM-06j force field45 for GlcNAc. Classical Force field parameters for UDP and m-Ins-1P (Supplementary Information, Figure S1) were generated with the Antechamber module of the AMBER suite, after geometry optimization and RESP charges derivation at the HF/6-31G* level with Gaussian g09, Revision D.0146. The Equilibration protocol consisted of 500-cycle runs of minimization with a 100 (kcal/mol)/Å2 restraint constant applied to the protein in order to relax the solvent structure, followed by a 2000-cycle energy optimization in which the restraint was removed to lose initial unfavorable contacts. The system was then slowly heated to 300 °K during a 200 ps simulation, with the Berendsen Thermostat. Finally, pressure was equilibrated at 1 atm over 500 ps, to let the system reach the proper density. All simulations used the periodic boundary condition approximation, the Ewald summation method with a 12 Å cut-off for non-bonded interactions, and the SHAKE algorithm was applied to all hydrogen-containing bonds. Final production 100 ns MD simulations were performed at 300 °K using the Langevin Thermostat and a 2 fs time step, from which the last structure was selected for QM-MM simulations. The 5F-GlcNAc inhibitor starting structure was generated by replacing the hydrogen atom 'H5' with fluorine on the topology file of the starting QM/MM structure, and carefully equilibrating it to let the moiety and its surroundings relax properly.

QM-MM Umbrella Sampling Simualtions The QM region consisted of 64 atoms (Fig. 2), including the whole GlcNAc donor, the diphosphate moiety of UDP leaving group, the m-Ins-1P acceptor without the phosphate group, and two H link atoms. It should be noted that, although partitioning the QM and MM subsystems in a polar bond (such as m-Ins-1P C1-O bond) is usually against general advice, we opted for this in order to avoid including the phosphate group in the QM subsystem. This inclusion would have required the incorporation of further sidechain functional groups bearing compensating charges, thus significantly increasing the QM subsystem size and consequently the computational cost. LIO is an implementation of DFT with Gaussian basis sets at the Generalized Gradient Approximation level, which works with the AMBER code setting up a QM/MM application. All relevant parts of the computation of LIO is ported to GPU, obtaining a great impact on performance, including exchange and correlation, Coulomb and the QM/MM coupling terms20,21. For this particular system under study, a common 1 ps long DFT QM/MM simulation on NVIDIA’s Tesla K20X GPUs could take up to 10 hours (~35 seconds per integration step, over a total of 1000 steps). All DFT QM/MM calculations in this work were performed with the Sander AMBER program and the LIO DFT program connected through Sander’s external QM/MM interface47, with the PBE functional using the DZVP basis. For umbrella sampling48 calculations, reaction coordinate (RC) was defined as the combination of the two distances that represent, respectively, the bond hydrolysis 'd1= d(C1-O3B)', and the bond formation at the anomeric center 'd2= d(O3-C1)'. The RC was sampled in increments of 0.1 Å intervals yielding a total of 48 sampling windows, using a 200 (kcal/mol)/Å2 force constant. Each window consisted in an initial 0.5 ps equilibration of the system, using always as starting structure the last snapshot from the previous window. Then, each window simulation was ACS Paragon Plus Environment

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extended for a least a further 6 ps, with the last 4 ps being used as production data. All simulations were carried with a 1 fs time step and a QM/MM electrostatic cutoff of 16 Å, at 300 °K (Langevin Thermostat) and 1 atm (Berendsen Barostat). The rest of the conditions were kept indentical as mentioned in the above section for the classical production MD simulations (a sample Sander input file is provided in Supplementary Information, Figure S2). Free Energy Profiles (FEP) were calculated employing the WHAM program49, dividing the sampled data into 100 bins and considering a convergence criterion of 0.0001. For comparative purposes, we also determined the FEP and reaction mechanism with lower and computationally faster levels of theory for the QM subsystems. Specifically, we tested the following Density Function Tight-Binding (DFTB) AMBER implementations50,51: DFTB2 (also known as SCC- DFTB), DFTB2-PA52 (which contains on-site third-order terms to improve proton affinities of phosphate compounds) and DFTB2-PR52 (a “reaction specific” improvement for phosphate hydrolysis reactions). DFTB2 parameters (Slater-Koster files) were retrieved from the dftb.org website. For all DFTB2 calculations the 'mio-1-1' parameter set was used.

Fig 2. Schematic representation of MshA's Active Site. Colored in Blue are the 64 atoms treated quantum-mechanically, the Red zigzag lines indicate where the 2 link atoms were placed. The main polar interactions involved in the anchoring of the reactants are represented with dashed lines, green for Hydrogen bonds and orange for salt bridges.


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RESULTS Stability of the complete MshA ternary-complex classical MD simulation was carefully inspected both visually and by means of plotting the Root Mean Squared Deviation (RMSD) as a function of time for all the protein heavy atoms (Supplementary Information, Figure S3). Results showed that the model was stable after a 100 ns run, so we could proceed with QM/MM calculations. To determine the free energy profile (FEP) and underlying mechanism of MshA, we applied an umbrella sampling strategy using as reaction coordinate the combination of the bond forming “m-Ins-1P O3 - GlcNAc C1” and bond breaking “GlcNAc C1 - β-phosphate O3B” distances. This RC allows the system not only to decide the order of events related to the bond-breaking and bond-formation on C1, but also allows HO3 proton transfer to occur unbiased. The corresponding FEP obtained at the DFT(PBE)-DZVP level is shown in Figure 3A, while key distances and Mulliken population plots are shown in Figures 3B and 3C, respectively. The profile shows that the reaction is exergonic by ca. 5 kcal/mol and presents a moderate 17.5 kcal/mol barrier. Analysis of the plots show that during the first stage of the reaction (RC between -2 and -1), O3 approaches the anomeric center up to ~ 2.7 Å (initial drop in d2). Afterwards, the key process leading to the Transition State (TS) clearly corresponds to the breaking of the “GlcNAc - β-phosphate” bond (d1), with the concomitant strengthening of the “β-phosphate O3B - m-Ins-1P HO3” hydrogen bond. Release of the diphosphate as the reaction trigger is also evidenced in the Mulliken analysis, which shows that the diphosphate leaving group becomes negatively charged. In the TS zone (Figure 3D, RC between 0 and 0.5), the most important geometrical change concerns the GlcNAc ring, which as expected instead of the initial 4 C1 chair shows a conformation that resembles more a 4 H5 half-chair, consistent with the formation of an oxocarbenium ion at C1. Further evidence for the ionic character of the TS comes from the C1 almost planar geometry (planar angle is >160° in the TS region) and the significant increase in the GlcNAc positive charge. Also important is the fact that in the TS the attacking nucleophile still has the proton attached, although it is partially shared with the diphosphate. The final stage of the reaction clearly depends on the concerted proton transfer from m-Ins-1P O3 to the β-phosphate (see d3 and d4) and formation of the O3-C1 bond (d2) and, lastly, complete detachment of the now protonated diphosphate. It is striking to observe the tight coupling between the bond formation and proton transfer, which occurs spontaneously, since -as already mentioned- the proton transfer is not part of the RC and thus no external force acts on it. Closer inspection of the substrates geometry in view of the observed mechanism also reveal two key interactions that help stabilize the building of negative charge on the UDP β-phosphate: First, donor GlcNAc acetamide group hydrogen bond, and second, acceptor m-Ins-1P hydroxyl group HO3 hydrogen bond. These interactions can be considered as a form of substrate assisted catalysis5,30.


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Fig 3. A) Free Energy Profiles as a function of the Reaction Coordinate, at the DFT(PBE)/DZVP level for the natural substrate (orange) and the fluorinated substrate analog (black). B) Distances as a function of RC for the main atoms involved in the reaction center C) Mulliken populations for the most relevant charged centers for natural substrate (full lines) and for fluorinated substrate analog (dotted lines) D) Transition State representative structure.

Overall, the results suggest a dissociative reaction, that can be assigned to a single-displacement concerted mechanism (DNAN), shown in Figure 1D, with the leaving group detachment as the key step, and a clear oxocabenium-ion-like character centered on GlcNAc C1. As for TS identification, it can be seen that there is a rather flat and wide energy region corresponding to oxocarbenium-ion-like structures with very similar energies. Altogether, these properties make MshA a very interesting case for understanding how catalytic efficiency is achieved in GTs (see discussion). To analyze MshA reaction further, and particularly in relation to possible inhibition mechanisms, we decided to study the reaction but using UDP-(5F)-GlcNAc instead as an alternative substrate/inhibitor. The corresponding FEP profile -computed with the same strategy and level of theory- is also shown in Figure 3A. The data shows why UDP-(5F)-GlcNAc apparently acts a competitive inhibitor. As expected, it binds exactly as the natural substrate, but despite the transfer reaction is still exergonic, the free energy barrier is over 30 kcal/mol resulting in an extremely slow reaction rate. Analysis of the underlying mechanistic steps (data not shown) evidence that for the fluorinated analogue mechanistic steps are the same as those previously described for the natural substrate, and therefore their different behaviour must result from subtle differences in how the TS state is reached and stabilized in each case.


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Detailed analysis of the FEP and distances and Mulliken Population plots show that the main difrerence bteween natural and fluorinated substrate concerns the free energy needed to reach the TS region, which requires breaking of the phosphate-sugar bond and developing of the oxocarbenium character in the GlcNAc. First noticeable difference between both substrates concerns the charge of the leaving diphosphate, which starts more negative in the natural substrate and draws charge earlier as the TS develop. This difference is clearly due to the presence of the fluorine attached to C5 because, while the initial charge in the C5-H(F) is about +0.2e in the natural substrate, it is only +0.1e in the fluorinated analogue. Moreover, when reaching the TS in the fluorinated analogue, the charge of the C5-F is significantly increased. Overall, the positive charge in the TS is similar in magnitude and distribution, but is reached earlier in the natural substrate. In other words, and as expected, the fluorine atom increases the free energy barrier since, due to its electronegativity, more energy is required to withdraw its charge allowing an optimal charge distribution across the whole GlcNAc moiety that promotes diphosphate leaving group to be easily released.

DISCUSSION Given their ubiquitous presence and relevance, glycosyltransferases reaction mechanisms have been extensively studied using theoretical and experimental chemistry methods. With the exception of GT-6 family members, for those retaining GTs which lack a putative residue that can act as a catalytic base a front-side SNi-like mechanism is generally proposed. This mechanism was first proposed in 1937 to elucidate unusual stereochemical outcomes of alkyl halides8. In the 1980s', it was re-introduced to explain the retention of the anomeric configuration stereochemistry in the solvolysis of α-glucosyl fluorides in mixtures of ethanol and trifluorethanol9, and was also used to explain the catalytic mechanism of an enzyme (glucogen phosphorilase) for the very first time10. The first crystallographic X-ray structure of a retaining Glycosyltransferase in complex with both complete donor and acceptor moieties was that of GT-A α-1,4-galactosyltransferase ‘LgtC’ from N. meningitidis, reported in 2001 by Persson et. al11. This authors were among the first to suggest that, given the absence of a putative active site base, a SNi-like mechanism with an "oxocarbenium-ion-like" transition state was the most probable scenario. A year later, Gibson et al. solved the structure of the first retaining GT-B, Trehalose-6-Phosphate Synthase ‘OtsA’ from E. coli, for which a similar mechanism was propposed53. Almost a decade after, separate groups carried for the first time full-enzyme QM/MM calculations on these structures. Despite using different strategies and sampling schemes, both groups were able to reproduce the SNi-like mechanism, but at the same time arrived at different results. Gomez et. al. work on LgtC25 consisted on both a Constrained Optimizations at the QM(DFT)/MM level to obtain a 'Potential Energy Profile' and also Umbrella Sampling at the SCC-DFTB level to obtain the corresponding 'Free Energy Profile'. Both results showed a rather concerted SNi-like mechanism with an oxocarbenium-ion-like Transition state (ANDN); Ardevol et. al. work on OtsA26, on the other hand, carried out Car-Parrinello DFT/PBE-based metadynamics simulations to construct a ‘Free Energy Landscape’ of the reaction, and their results suggested a step-wise SNi-like mechanism (DN*ANss), in which a short-lived oxocarbenium-phosphate ion pair intermediate was formed. Kinetic isotope labeling and linear free-energy relationship investigation conducted the same year on OtsA by Seo-Lee et. al.54 . gave experimental evidence and further support to the single-displacement SNi-like mechanism model, but were unable to confirm whether the reaction proceeds via a stepwise or a concerted mechanism. After this two pioneer works on full-enzyme models of retaining GTs, many other cases were studied, becoming an active field of research. Our results, performed for the metal-independent retaining GT-B MshA of Corynebacterium glutamicum at the QM(DFT-PBE)/MM level of theory using the umbrella sampling strategy suggest a concerted and highly dissociative SNi-like mechanism (DNAN), similar to that observed ACS Paragon Plus Environment

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for LgtC, but with a rather flat high-energy region which makes it difficult for a precise TS structure identification. Also important is that in both cases proton transfer occurs late in the reaction. The obtained activation energy barrier of 17.5 kcal/mol, is also in remarkable agreement with the experimental data from Vetting et. al., who determined a Kcat of 12.5 s-1 from steady state kinetics35, which can be assigned to a ~17.02 kcal/mol barrier using transition state theory and the Eyring equation, and in the range of previous reported value of 23 kcal/kmol for the above cited metal-independent GT-B OtsA in silico metadynamics study26. Furthermore, we also determined the FEP and underlying mechanism for the reaction of the fluorinated substrate analogue, previously reported as a competitive inhibitor40. Consistent with the experimental data, we obtained a significantly higher barrier (33 kcal/mol), which would result in a very slow reaction and thus explain the experimentally observed inhibition. Detailed analysis of the fluorinated analogue behaviour shows that the most likely reason for the increase in the barrier is the electronegativity of the fluorine atom that impedes charge transfer to the leaving phosphate, which is further consistent with a dissociative concerted mechanism and oxocarbenium-ion-like TS. An important aspect of this work concerns the level of theory used. Given the high computational cost of QM(DFT-PBE)/MM (or similar functionals, like B3LYP), and that significantly high free energy barriers were reported at the SCC-DFTB level for GTs LgtC (32.7 kcal/mol)25 and EXTL2 (40.5 kcal/mol)32, it is important to assess their gain in accuracy compared to faster simplified methods. Figure 4 shows the FEP for MshA catalyzed reaction obtained with different Semiempirical methods, namely DFTB2, DFTB-PA and DFTB-PR (see methods for details). The results for the different DFTB approximations show that all display higher barriers (up to 45 kcal/mol) and that there are very large differences among them. The lowest barrier is obtained with the DFTB-PR scheme (~ 25 kcal/mol), the one that has been specifically adjusted for phosphate transfer reactions, which again hints this event as the key step for the system under study. Detailed analysis of the underlying mechanism also shows that in all DFTB versions, proton transfer occurs immediately after TS formation. Although further analysis of the reasons for these observations are beyond the scope of this publication, we suggest possible failure of DFTB methods to stabilize dissociative ion-par like intermediates.


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Fig 4. Free Energy Profiles at various DFTB levels.

Another interesting point concerns the computational method used. It should be noted that classic Umbrella Sampling MD, as well as other free-energy based approaches like metadynamics (dynamic QM/MM), are conceptually different and computationally more expensive than the static approaches based on geometrical restrained optimizations along a reaction coordinate, in which thermal effects on atomic motion and entropy are neglected and ‘potential energy’ is calculated instead of ‘free energy’. Having had a vast experience with static methods in the past14-17, we are strong supporters of dynamic methods, since we are inclined to believe dynamic motions of proteins are strongly related to their function. In this context, it is interesting to note that very recently, three different groups all conducted in silico studies on the same metal-dependent GT-A, the ‘polypeptide UDP-GalNAc transferase 2’ (or “ppGalNAcT2”). One of them used metadynamics and obtained a step-wise mechanism27, while the other two used a static approach, with one obtaining a step-wise29 and the other a concerted mechanism but with no identifiable TS due to a rather flat high-energy region28. Facing such a scenario, and although it is still difficult to give a definitive answer, the fact that both static and dynamic methods have rendered both types of mechanism for the same GT-A (ppGalNAcT2), and that now two dynamic methods have yielded both mechanisms for two similar GT-Bs' (OtsA and MshA), suggest that both mechanisms might be feasible. As in almost any simulation method, there is a tradeoff between accuracy and sampling, given a certain available computational time. In this work we have decided to increase the method accuracy, using DFT quantum mechanics accelerated by GPUs, which allowed us to do roughly 6 ps per umbrella window. While this might seems like a short time, it is important to realize that without the use of GPUs, even 6 ps would be completely out of reach. The 6 ps time is in fact applied to the degrees of freedom orthogonal to the reaction coordinate, so it mostly reflects the relaxation of the local enviroment to changes in electron density during the reaction. We already know that these changes are localized and not large, which means that they are reasonably sampled in this time scale. If the system were to undergo very large conformational changes, ACS Paragon Plus Environment

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then of course 6 ps would not be enough and an alternative would be needed. As a final remark on the subject, although strong supporters of dynamic methods, we are also well aware that simulation times achievable at QM(DFT)/MM level are still quite limited by the high computational cost they demand and, thus, there is still a lot of contribute to the field in order to obtain shorter times that enable the user to have more sampling of the configurational space.

CONCLUSIONS In summary, our results show that Corynebacterium glutamicum MshA reaction occurs through a concerted dissociative SN i-like mechanism (DNAN), with a flat high-energy region of oxocarbenium-ion-like structures representative of the Transition State. Our data also suggest that the unstable dissociative nature of this TS requires real DFT level of theory to be accurately described, and tight binding methods fail to describe accurately both the underlying molecular events and the resulting free energy along the reaction. Analysis of fluorinated substrate analogue shows that it acts as an inhibitor due to a significant increase in the reaction activation energy barrier, motivated by the fluorine reluctance to transfer electron density to the leaving group.

SUPPORTING INFORMATION AVAILABLE Supporting Information includes classical force-field parameter files in AMBER-compatible format for both MshA substrates, UDP and myo-inositol-1-Phosphate. We also provide a sample Sander input file to run a typical QM/MM Umbrella Sampling window simulation using the external module LIO with AMBER. Finally, the Root Mean Square deviation of all protein heavy atoms as a function of time (step) for the classical MD simulation of the MshA ternary complex is attached.

ACKNOWLEDGEMENTS This work was primarily funded by grants CONICET- PIP 112 201101 00850, PICTO-2012-0057 and RAICES-Siembra 2014-2015 awarded to Marcelo A Martí. Marcelo A. Martí is a member of CONICET. The simulations were conducted with the Blue Waters supercomputer, supported by NSF Grants ACI-0725070 and ACI-1238993, and the Extreme Science and Engineering Discovery Environment (XSEDE), supported by NSF Grant OCI-1053575. The work in Blue Waters was supported by the NSF-PRAC allocation 1440031 to AER. We would also like to thank the High-Performance Computing Center at the University of Florida.

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Theoretical Insights into the Reaction and Inhibition Mechanism of

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